Systems and Methods for Anti-PAX8 Antibodies

ABSTRACT

The present invention is related to the anti-PAX8 antibodies, kits, cocktails, and use of anti-PAX8 antibodies for detection of cancer.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.14/507,667 filed Oct. 6, 2014, which is a continuation of U.S.application Ser. No. 14/004,400 filed Sep. 19, 2013, now issued as U.S.Pat. No. 8,852,592, which is the U.S. National Phase of InternationalPatent Application No. PCT/US2012/037367 filed May 10, 2012 which claimspriority to and the benefit of U.S. Provisional Application No.61/484,579 filed May 10, 2011 and U.S. Provisional Application No.61/588,035 filed Jan. 18, 2012, each application hereby incorporated byreference herein in their entirety.

TECHNICAL FIELD

This invention relates to novel PAX8 antibodies, compositions, and kitscomprising the antibodies and methods for using the antibodies.

BACKGROUND OF THE INVENTION

Microscopic examination of tissue samples, particularly those obtainedby biopsy, is a common method for diagnosis of disease. In particular,immunohistochemistry (IHC), a technique in which specific antibodies areused to detect expression of specific proteins in the tissue sample, isa valuable tool for diagnosis, particularly for the detection anddiagnosis of cancer.

The paired box (PAX) genes are a family of cell-lineage transcriptionfactors that may play fundamental roles during organogenesis and areregulatory proteins expressed in normal and neoplastic cells of the samelineage. PAX8 is a nephric-lineage transcription factor that may be acrucial transcription factor for organogenesis of the thyroid gland,kidney and Müllerian system. These proteins are required for cell growthand differentiation in embryonic tissues and can be expressed in adulttissues and in specific cell-lineage neoplastic tissues.

PAX8 may have been shown to be a useful marker of several cancers,particularly kidney, ovarian, endometrial, and thyroid cancers.Detection of PAX8 by anti-PAX8 antibodies, using immunohistochemistry,may have been shown to be a valuable tool for detection and diagnosis ofthese cancers.

Immunohistochemical detection of PAX8 expression may be advantageous forseveral reasons: PAX8 may be present in a high percentage of cases ofthese cancers; PAX8 can identify both primary and metastatic tumors ofthese types; and even nuclear expression of PAX8 may result in strongstaining of the nucleus, which may ease interpretation and diagnosis.

Unfortunately, some known anti-PAX8 antibodies useful forimmunohistochemistry may have the disadvantage that they cross-reactwith lymphocytes, particularly B-cells, which may frequently infiltrateinto the site of a tumor. Simultaneous staining of B-cells, alongsidepositive staining of PAX8, can complicate analysis and eveninterpretation of the tissue sample. In such cases, the pathologist mayhave to rely on other methods (e.g., morphological differences) todiscriminate B-cell staining from tumor cells. Furthermore, staining ofB-cells can be a significant disadvantage in the analysis of tissuesamples from lymph nodes, a common scenario when evaluating thepotential of metastasis into a lymph node. Considering that lymph nodesmay naturally contain large numbers of B-cells, staining a lymph nodesample with one of the currently known PAX8 antibodies may result inextensive staining of B-cells and may make identification of metastatictumor cells in the lymph node extremely difficult. A more specificanti-PAX8 antibody that does not cross-react and stain B-cells couldoffer a significant advantage by simplifying interpretation, resultingin clearer, more confident and even accurate diagnosis.

DISCLOSURE OF THE INVENTION

General embodiments of the present invention may include monoclonalantibodies for recognizing PAX8, methods for their preparation, use inimmunohistochemistry, and the like. This mouse monoclonal anti-PAX8antibody [BC12] may be useful for the detection of PAX8 in tissuesamples, perhaps with several significant, but unexpected advantagesover currently known PAX8 antibodies. When used in traditionalimmunohistochemistry procedures, the mouse PAX8 antibody [BC12] mayresult in nuclear staining of PAX8 with a sensitivity perhaps similar tothat of known PAX8 antibodies. However, BC12 may exhibit increasedspecificity, perhaps as compared to past PAX8 antibodies, which mayoffer significant improvements. In contrast to known PAX8 antibodies,the present invention's mouse PAX8 antibody [BC12] may not stainB-cells. As a result, BC12 may offer a considerable advantage fordiagnosis, since the interpretation of the sample may not be complicatedby staining of infiltrating B-cells. With BC12, analysis of the samplemay be simplified and PAX8 expression in tumor cells may be readilyidentifiable. Furthermore, evaluating the presence of metastatic tumorcells in lymph node samples may be uncomplicated and straightforwardwhen using BC12, as BC12 may avoid the complications associated withstaining of the B-cells that may be naturally present in the lymph node.

Furthermore, BC12 may not stain normal or neoplastic pancreatic tissueand neuroendocrine cells in normal stomach. A common feature of pastPAX8 antibodies may be the staining of normal pancreatic tissue and somecases of pancreatic cancer and in some cases, neuroendocrine cells inthe stomach. Such additional reactivity and lack of specificity, canlead to ambiguity in diagnosis, particularly in cases of metastaticdisease, where the primary tumor may be unknown. The increasedspecificity of BC12 resulting from the lack of cross-reactivity andstaining of pancreatic tissue and certain neuroendocrine cells may be anadvantage for its use, leading to less ambiguous, more confidentdiagnosis.

Naturally, further objects, goals and embodiments of the invention(s)are disclosed throughout other areas of the specification, claims, anddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of PAX8 staining on kidney tissue with PAX8Mouse Monoclonal [BC12] at 20×.

FIG. 2 shows an example of PAX8 staining on kidney tissue with PAX8Rabbit Polyclonal at 20×.

FIG. 3 shows an example of PAX8 staining on Ovarian Cancer tissue withPAX8 Mouse Monoclonal [BC12] at 10×.

FIG. 4 shows an example of PAX8 staining on Ovarian Cancer tissue withPAX8 Rabbit Polyclonal at 10×.

FIG. 5 shows an example of PAX8 staining on a Clear Cell Renal Carcinomawith PAX8 Mouse Monoclonal [BC12] at 20×.

FIG. 6 shows an example of PAX8 staining using PAX8 Mouse Monoclonal[BC12] on Tonsil at 10× and having no stain on the B-cells.

FIG. 7 shows an example of PAX8 staining using PAX8 Mouse Monoclonal

[BC12] on Tonsil at 20× and having no stain on the B-cells.

FIG. 8 shows an example of PAX8 staining using PAX8 Rabbit Polyclonal onTonsil at 10× wherein staining of B-cells is present.

FIG. 9 shows an example of PAX8 staining using PAX8 Rabbit Polyclonal onTonsil cells at 20× wherein staining of B-cells is present.

FIG. 10 shows an example of PAX8 staining using PAX8 Mouse Monoclonal[BC12] on pancreas tissue at 10× wherein no stain is present.

FIG. 11 shows an example of PAX8 staining using PAX8 Mouse Monoclonal[BC12] on pancreas tissue at 20× wherein no stain is present.

FIG. 12 shows an example of PAX8 staining using PAX8 Rabbit Polyclonalon pancreas tissue at 10× wherein stain is present.

FIG. 13 shows an example of PAX8 staining using PAX8 Rabbit Polyclonalon pancreas tissue (islets of Langerhans) at 20× wherein stain ispresent.

FIG. 14 shows an example of PAX8 staining using PAX8 Mouse Monoclonal[BC12] on stomach tissue at 20× wherein no stain is present.

FIG. 15 shows an example of PAX8 staining using PAX8 Rabbit Polyclonalon stomach tissue at 20× wherein stain is present. Note: A chromograninIHC stain confirmed that the rabbit PAX8 staining was of neuroendocrineorigin.

FIG. 16 shows an example of staining of using PAX8+p63 antibody cocktailon renal cell carcinoma.

FIG. 17 shows an example of staining of using PAX8+p63 antibody cocktailon normal bladder.

FIG. 18 shows an example of staining of using PAX8+PSA+GATA-3 antibodycocktail on renal cell carcinoma.

FIG. 19 shows an example of staining of using PAX8+PSA+GATA-3 antibodycocktail on prostate adenocarcinoma.

FIG. 20 shows an example of staining of using PAX8+PSA+GATA-3 antibodycocktail on urothelial carcinoma.

FIG. 21 shows an example of staining of using PAX8+ER+mammaglobincocktail on renal cell carcinoma.

FIG. 22 shows an example of staining of using PAX8+ER+mammaglobincocktail on normal breast.

FIG. 23 shows an example of staining of using PAX8+NKX3.1 antibodycocktail on renal cell carcinoma.

FIG. 24 shows an example of staining of using PAX8+NKX3.1 antibodycocktail on prostate adenocarcinoma.

FIG. 25 shows an example of staining of using PAX8+Napsin A antibodycocktail on renal cell carcinoma.

FIG. 26 shows an example of staining of using PAX8+Napsin A antibodycocktail on normal lung.

FIG. 27 shows an example of staining of using PAX8+CK20 antibodycocktail on renal cell carcinoma.

FIG. 28 shows an example of staining of using PAX8+CK20 antibodycocktail on normal colon.

FIG. 29 shows an example of staining of using PAX8+ERG+GATA-3 antibodycocktail on renal cell carcinoma.

FIG. 30 shows an example of staining of using PAX8+ERG+GATA-3 antibodycocktail on prostatic adenocarcinoma.

FIG. 31 shows an example of staining of using PAX8+ERG+GATA-3 antibodycocktail on urothelial carcinoma.

FIG. 32 shows an example of a schematic summary of a kit in accordancewith various embodiments of the present invention.

FIG. 33 shows an example of a schematic summary of an immunoassay methodin accordance with various embodiments of the present invention.

MODE(S) FOR CARRYING OUT THE INVENTION

As may be understood from the earlier discussion, the present inventionincludes a variety of aspects, which may be combined in different ways.The following descriptions are provided to list elements and describesome of the embodiments of the present invention. These elements arelisted with initial embodiments, however it should be understood thatthey may be combined in any manner and in any number to createadditional embodiments. The variously described examples and preferredembodiments should not be construed to limit the present invention toonly the explicitly described systems, techniques, and applications.Further, this description should be understood to support and encompassdescriptions and claims of all the various embodiments, systems,techniques, methods, devices, and applications with any number of thedisclosed elements, with each element alone, and also with any and allvarious permutations and combinations of all elements in this or anysubsequent application.

Embodiments of the present invention may provide antibodies, monoclonalantibodies, any fragments thereof such as antigen binding fragmentsthereof, and methods thereof that specifically bind to PAX8 and may beused for the detection of PAX8 in the detection, diagnosis, prognosis,prediction of outcome of treatment, assessment of efficacy, assessmentof recurrence, or the like for several types of cancers. The monoclonalantibody may be an antibody fragment, a mouse monoclonal antibody, ahumanized monoclonal antibody, a human monoclonal antibody, an antibodyconjugated with a label, an antibody labeled with a detectable signal orstain, an antibody labeled with a toxin, or the like. Examples of labelsmay include but are not limited to radioactive element, magneticparticles, radioisotope, fluorescent dye, enzyme, toxin, signal, stain,any combination thereof, or the like. Systems and methods of the presentinvention may relate to the monoclonal antibody or its antigen bindingportion capable of binding to PAX8. In other embodiments, the presentinvention may provide a monoclonal antibody or its antigen bindingportion thereof capable of binding to PAX8 but which does not bind toB-cells.

Mouse monoclonal antibodies may be commonly used in immunoassay methodsto identify specific analytes, including as primary antibodies inimmunohistochemistry procedures. Mouse monoclonal antibodies specificfor the protein target of interest can typically be produced usinggenerally known procedures. Generally, exposing a mouse to the antigenof interest (e.g. a peptide fragment of the desired target or thefull-length protein target) may induce an immune response in which themouse generates multiple antibodies that bind the antigen, each of whichmay be produced by a particular B-cell. These B-cells may be isolatedfrom the mouse spleen and the antibodies produced may be evaluated fortheir suitability as primary antibodies in IHC. After selecting theoptimal antibody, the associated B-cell may be fused with a tumor cellusing known procedures, perhaps resulting in a hybridoma, a new cellline that can endlessly replicate and may continuously produce thedesired antibody.

Monoclonal antibodies may be preferred over polyclonal antibodies forseveral reasons. In particular, monoclonal antibodies may be derivedfrom a single B-cell and as such may recognize a single epitope, perhapsresulting in greater specificity. Monoclonal antibodies may also beconveniently and reproducibly generated in cell culture, perhapsresulting in a constant supply of the desired antibody. In embodiments amonoclonal antibody may include but is not limited to an isolatedmonoclonal antibody, a mouse monoclonal antibody, a rabbit monoclonalantibody, a goat monoclonal antibody, a horse monoclonal antibody, achicken monoclonal antibody, a humanized monoclonal antibody, a chimericantibody, and any combination thereof, or the like.

Embodiments of the present invention may provide a kit (5) and methodsof using such kit which may be a diagnostic or prognostic kit thatincludes an antibody or fragment thereof or even portion thereof asdiscussed herein with perhaps an antibody detection element of theantibody, fragment, or portion thereof when bound to an antigen where abiological sample (2) may be contacted with the antibody, fragment, orportion thereof and detection of the bound antibody-antigen may bedetermined. A biological sample may include but is not limited to anormal tissue, neoplastic tissue, kidney tissue, ovarian tissue, thyroidtissue, endometrial tissue, renal tissue, tonsil tissue, pancreastissue, colon tissue, lymph node tissue, neoplastic pancreatic tissue,stomach tissue, bladder tissue, prostate tissue, lung tissue, breasttissue, or the like. As discussed herein, use of the antibody orfragment thereof or portion thereof or composition may performed on anautomated staining device such as with methods including but not limitedto immunoassay, immunohistochemistry (IHC), IHC of FFPE, ICH offrozen-tissue sections, and ELISA. In embodiments, detection of theantibody-antigen binding may be made manually, automatically, via imageanalysis or the like and may even be made via an automated stainingdevice.

As but one example of an immunoassay method, embodiments of the presentinvention may provide obtaining tissue from an animal or human to betested (6), fixing or freezing said tissue (7), treating said fixed orfrozen tissue to unmask epitopes to PAX8 (8), contacting said treatedtissue with an antibody or fragment thereof as discussed herein in anamount and under conditions such that said antibody or fragment thereofbinds to a PAX8 protein if said protein is present in said tissue (9);and perhaps even detecting the presences of said bound antibodies (10),as schematically represented in FIG. 33.

FIG. 32 shows a schematic summary of various embodiments of the presentinvention including a kit (5) which may provide an antibody, fragmentthereof, portion thereof, in a composition or even in a cocktail (1),perhaps even provided from a hybridoma, the antibody (1) or the like maybe contact with a biological sample (2) to form at least oneantibody-antigen complex (3) which may then be detected with a detector(4).

In embodiments, the PAX8 antibody clone BC12 can be obtained byimmunizing Balb/C mice with a full length human PAX8 recombinant proteinobtained by E. coli expression. The PAX8 proteins may be injected intothe BALB/c mice, with an adjuvant, via subcutaneous and intraperitonealinjections alternatively, about 5 times at about three week intervals.The immune reactivity to PAX8 may be assessed by direct ELISA onrecombinant PAX8 protein. Mice with the highest titer may be chosen fordeveloping hybridomas by cell fusion. A hybridoma clone demonstratingthe best reactivity to PAX8 on human tissues may be chosen and may bedesignated as BC12. The BC12 clone may be tested for isotype and may beidentified as a mouse IgG1/kappa. The BC12 antibody may be produced bylarge scale tissue culture of the hybridoma cells and by ascites inBALB/c mice. The supernatant and antibody ascites may be collected andthe antibody may be purified by Protein A affinity column. BC12demonstrated specific reactivates to human PAX8 protein by ELISA,Western blotting, and even human tissues.

Mouse monoclonal anti-PAX8 antibody [BC12] may be produced using thesegeneral procedures and may be evaluated by immunohistochemistry forsensitivity and specificity on a variety of normal and neoplastictissues, particularly in comparison to the previously known rabbitpolyclonal PAX8 antibody that is widely used.

Example of PAX8 protein expression: A full-length PAX8 recombinantprotein may be cloned and expressed from E. Coli. Briefly, PAX8 cDNA maybe cloned and purified. The PAX8 cDNA may be digested by restrictionenzymes and ligated into the pET30a-GST vector. BL21 cells may betransformed with the construct. The colonies expressing the correct sizeof recombinant protein may be selected and sequenced. A further scale upproduction may be performed by culturing the E. coli in LB mediacontaining 0.5 mM IPTG. The final PAX8 recombinant protein may bepurified and analyzed by SDS-PAGE.

Example of Host Immunization: Female BALB/c (about 6 to about 8 weeksold) mice may be immunized intraperitoneally (i.p.) with about 100 μghuman PAX8 protein per mouse in complete Freund's adjuvant. About threeweeks later, the mice may be boosted with another 100 μg human PAX8 permouse in incomplete Freund's adjuvant about 4 more times in about 3 weekintervals. Mice may be bled from the tails, and sera may be collectedand stored at −20° C. for later analysis of antibody titers byenzyme-linked immunosorbent assay (ELISA).

Example of Hybridomas: Hybridomas producing antibodies to PAX8 may begenerated by standard techniques from splenocytes of PAX8-immunizedBALB/c mice. Briefly, splenocytes from PAX8-immunized mice may be fusedto P3-X63-Ag 8.653 myeloma cells (non-secreting myeloma derived fromSP2/0 Balb/c myeloma cells) by incubation with about 50% polyethyleneglycol at a ratio of about 4:1. Following incubation, cells may bepelleted by centrifugation at about 3000×g for about 10 minutes, washedin about 25 ml of PBS, recentrifuged, and cell pellet may be resuspendedin about 100 ml of fresh Dulbecco's Medium containing about 20% fetalbovine serum (Hyclone, Utah, Co). Aliquots of about 100 μl can be addedto each well of ten 96-well microtiter plates (Corning, Lowell, Mass.).About twenty four hours later, about 100 μl DMEM culture mediumsupplemented with about 1M hypoxanthine (HT), about 4 mM aminopterin andabout 160 mM thymidine (HAT) can be added to each microtiter well. Mediamay be replaced after about 4 days with complete media (perhapscontaining HAT and HT). Over the following about 10 days, media may beremoved and replaced with fresh media with reduced or perhaps even noHAT and HT added. Hybridoma supernatants may be screened by ELISA forantibody reactivity to PAX8, and hybridoma clones may then be selectedand stabilized by cloning twice by limiting dilution.

Hybridoma cells referred to as Anti PAX8 Mouse hybridoma clone BC12 Lot:042811 have been deposited at American Type Culture Collection (ATCC) inManassas, Va. on May 4, 2011 and the he deposit received ATCC PatentDeposit Designation No. PTA-11873.

ELISA: Host anti-sera immune responses to PAX8 may be measured by ELISA.Briefly, a solution of PAX8 (1 μg/ml) in phosphate-buffered saline (PBS)may be used to coat 96-well flat bottom polystyrene plates. The platesmay then be blocked with about 1% bovine serum albumin (BSA)-PBS. Eitherdiluted immune sera or hybridoma supernatants may be added and incubatedat about 37° C. for about 1 hour. After washing the plates with PBS, theplates may be incubated with goat anti mouse-HRP reagents (JacksonLabs). Incubations may be done at about 37° C. for about 30 minutes.ABTS substrate may be added to develop color and the absorbance at about405 nm (A405) may be measured in a microtiter plate reader.

Isotype of monoclonal antibodies: The BC12 monoclonal antibody may beisotyped using a mouse monoclonal antibody isotyping kit (Invitrogen,Carlsbad Calif.). Briefly, about 100 μl of supernatant from mousemonoclonal antibody [BC12] cells may be added to the plate coated goatanti mouse IgG1, IgG2A, IgG2B, IgG3, IgM, and IgA. After about 30minutes incubation, the plate may be washed 3 times with PBS and may beincubated with goat anti mouse Ig-HRP reagent. ABTS substrate may beadded to develop color and the absorbance at about 405 nm (A405) may bemeasured in a microtiter plate reader.

Antibody Production and purification: The selected hybridoma cells fromclone BC12 may be cultured with DMEM culture medium supplemented withabout 10% FBS. The culture supernatants may be further purified byprotein A affinity column. The hybridoma cells may also be injected intopristane-primed BALB/c mice to produce antibody ascites. The antibodyascites may be further purified by protein A affinity column. IgGconcentration may be measured spectrophotometrically using theextinction coefficient for human IgG of about 1.4 (about 0.1% at about280 nm). The purity of IgG may be determined by SDS-PAGE.

Western Blotting: The purified monoclonal antibody [BC12] may becharacterized by Western Blotting. Whole-cell lysates may be generatedfrom OVCAR3, HEK293 cells with lysis buffer (about 1% NP40, about 0.5%sodium deoxycholate, and about 0.1% SDS in PBS) in the presence ofprotease inhibitors. Lysate (between about 20 and about 30 μg/lane) wassubjected to protein gel electrophoresis using about 4 to about 12%SDS-PAGE with Tris-glycine buffer and may be transferred ontonitrocellulose filters in Tris-glycine buffer. Proteins on the blots maybe visualized by incubating BC12 antibody for about 60 minutes in roomtemperature after blocking with blocking buffer, followed by incubatingwith peroxidase-conjugated goat anti-mouse immnoglobulins.

Determination of VH and VL sequences: Total RNA may be extracted fromhybridomas using Qiagen kit (USA, Gaithersburg, Md.) as per themanufacturer's instructions. First-round RT-PCR may be carried out withQIAGEN® OneStep RT-PCR Kit. RT-PCR may be performed with primer setsspecific for the heavy and light chains. For each RNA sample, about 12individual heavy chain and about 11 light chain RT-PCR reactions can beset up using degenerate forward primer mixtures covering the leadersequences of variable regions. Reverse primers may be located in theconstant regions of heavy and light chains. No restriction sites may beengineered into the primers. The RT-PCR products from the first-roundreactions may be amplified in the second-round PCR. About 12 individualheavy chain and about 11 light chain RT-PCR reactions can be set upusing semi-nested primer sets specific for antibody variable regions.The amplified cDNAs can be gel purified and may then be sequenced.

[BC12] Variable Domains were sequenced to provide isolatedpolynucleotides that comprise nucleic acid sequences encoding the aminoacid sequences of one or more of the CDRs of the light and/or heavychain variable regions of a monoclonal antibody described herein thatbinds to the PAX8 EQGLYPLPLLNSTLD epitope. The sequence of the variableregion of the heavy chain is identified as SEQ ID NO: 1 and the sequenceof the variable region of the light chain is identified as SEQ ID NO: 2

Therefore, embodiments of the present invention may provide a hybridoma,antibodies or fragments thereof or even portions thereof produced by thehybridoma deposited with the ATCC under ATCC Patent Deposit DesignationNo. PTA-11873. As discussed herein, a method for producing a monoclonalantibody from the hybridoma may include culturing a hybridoma whichproduces a monoclonal antibody capable of specifically recognizing PAX8;and perhaps even allowing said hybridoma to produce the monoclonalantibody. Other embodiments may include providing an antibody which hasa binding specificity of PAX8 and which does not bind to B-cells.Alternatively, an antibody or fragment thereof may have a polypeptide ofthe amino acid sequence encoded by the nucleic acid sequence of SEQ IDNO: 1 and/or SEQ ID NO: 2. Embodiments may include an antibody orfragment thereof that specifically binds to at least one polypeptidewith an amino acid sequence of SEQ ID NO:3. Even yet, an antibody mayinclude an amino acid sequence which may be at least about 70% identicalto an amino acid sequence encoded by a nucleic acid sequence of SEQ IDNO: 1 and/or SEQ ID NO: 2. In other embodiments, an isolated andpurified nucleic acid sequence may include a nucleic acid sequence thatmay be at least about 70% identical to SEQ ID NO: 1 and/or SEQ ID NO: 2.Alternatively, an antibody or fragment thereof may be provided thatspecifically binds to at least one polypeptide with an amino acidsequence that may be at least 70% identical to residues of SEQ ID NO: 3This may provide that either SEQ ID NO:1, SEQ ID NO:2, or perhaps evenSEQ ID NO:3 may be modified in some way (e.g., change a few residues orthe like). The amount of modifications may vary depending on whatmodifications are made and perhaps even how the modified sequenceperforms. Other modifications may include: at least about 71%, at leastabout 72%, at least about 73%, at least about 74%, at least about 75%,at least about 76%, at least about 77%, at least about 78%, at leastabout 79%, at least about 80%, at least about 81%, at least about 82%,at least about 83%, at least about 84%, at least about 85%, at leastabout 86%, at least about 87%, at least about 88%, at least about 89%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, and at least about 99%.

The sequences of the variable regions of the heavy chain and light chaincan be computed using known software to one skilled in the art togenerate the complementarity determining regions (CDRs). Therefore, thesequence of the variable region of the heavy chain, SEQ ID NO:1, resultsin CDR sequences: SEQ ID NO: 4 (CDR1), SEQ ID NO: 5 (CDR2) and SEQ IDNO: 6 (CDR3). The sequence of the variable region of the light chain,SEQ ID NO:2 results in CDR sequences: SEQ ID NO: 7 (CDR1), SEQ ID NO: 8(CDR2) and SEQ ID NO: 9 (CDR3).

Epitope Mapping of the mouse anti-PAX8 [BC12] Binding Sequence: In orderto determine the peptide sequence of PAX8 that is recognized by [BC12],epitope mapping was conducted using two assays: direct ELISA and dotblot. In an ELISA assay, the sensitivity and specificity of theanti-PAX8 [BC12] antibody was determined by measuring the antibody titerat 1:500 and 1:1000. Overlapping peptides at a length of 15 amino acidseach, covering the full length of the human PAX8 protein, were used todetermine the preferred sequence of [BC12] binding. The anti-PAX8 [BC12]binds specifically to a peptide corresponding to residues 258-272 ofPAX8, which is EQGLYPLPLLNSTLD. The result was further confirmed by dotblot assay.

For direct ELISA protocol, the plates were first coated with 100 μl ofPAX8 peptides at 5 μg/mL in coating buffer (pH 9.5) overnight at 4° C.,followed by blocking (3% BSA) at 200 μl/well for 1 hour at roomtemperature. The plates were incubated with purified PAX8 antibody at100 ng/mL and 200 ng/mL separately for 1 hour at room temperature on anELISA-plate shaker. Then the plates were washed five times with PBST(300 μl/well) followed by the addition of goat anti-mouse IgG-HRP to theplates and incubation for 1 hour on a plate-shaker. The plates were thenwashed with PBST (300 μl/well) and blotted to dry, and TMB was added at100 μl/well, developed for 5 min on a shaker, followed by a stopsolution (50 μl/well). Absorbance was measured at 450 nm on an ELISAplate reader according to the manufacturer's recommendation.

For the dot blot assay, a nitrocellulose membrane was blotted with 1 μlat a concentration of 1 mg/ml the peptide, quadruplicates per peptide.This membrane was incubated for 1 hour at room temperature until it wascompletely dry. The membrane was blocked with 3% BSA in TBST (50 mMTris, 0.5 M NaCl, 0.05% Tween-20, pH 7.4) for 1 hour at roomtemperature, then mouse anti PAX8 antibody [BC12] was added at 200 ng/mlfor 1 hr at RT in TBST. Then the membrane was washed for 3 times (10minutes each) in TBST on an orbital shaker, followed by incubating withsecondary antibody goat anti mouse IgG1-AP for 1 hour at roomtemperature in TBST. The membrane was washed 3 times (10 minutes each)in TBST on a rocker. The binding was detected by adding Western GloChemiluminescent detection reagents and exposing to film.

IHC method with anti-PAX8 BC12: Immunohistochemistry using the mousemonoclonal PAX8 antibody [BC 12] may be performed on formalin-fixedparaffin embedded (FFPE) tissue samples using procedures generally knownto those in the art, as generally exemplified by the followingnon-limiting examples (washes with Tris-buffered saline, pH about 7.6,between steps):

1) Sections (˜5 m) of formalin fixed paraffin-embedded tissues may bemounted on commercially available microscope slides coated withpolylysine.

2) Sections may be deparaffinized (using xylenes or a xylene-substitute)and may be rehydrated through a series of alcohol/water solutions,followed by blocking of endogenous peroxidases with about 3% hydrogenperoxide solution.

3) Samples may be subjected to heat-induced antigen retrieval using acitrate buffer in a pressure cooker (Reveal, Decloaking Chamber; BiocareMedical) and may be heated to about 125° C. for about 30 seconds. [Otherantigen retrieval methods known to those skilled in the art (e.g.,steamer, microwave oven, and enzyme) may also be acceptable.] Tissuesmay be allowed to cool for about 10 minutes and then may be rinsed withdeionized water.

4) The PAX8 antibody [BC12] may be applied in a Tris-buffered solution(pH about 6.2) with bovine serum albumin as carrier protein for about 30minutes.

5) Detection of the PAX8 antibody with a horseradish peroxidase (HRP)conjugated secondary antibody (MACH 4 Universal HRP-Polymer Detection,Biocare Medical) may be accomplished in two steps. An initialapplication of a rabbit anti-mouse IGg antibody for about 10 minutes maybe followed by incubation with a goat anti-rabbit-HRP conjugate forabout 10 minutes.

6) In a final detection step, 3,3′-diaminobenzidine (DAB) in buffercontaining about 0.02% hydrogen peroxide (Betazoid DAB, Biocare Medical)may be applied. The oxidation of DAB through an HRP-mediated mechanismmay result in precipitation of a brown, chromogenic product, perhapsallowing identification of sites of PAX8 expression.

7) Slides may be briefly counterstained in a modified Mayer'shematoxylin.

Results of IHC Staining with mouse monoclonal anti-PAX8 antibody [BC12]:Using the above protocol, a variety of normal and neoplastic tissueswere evaluated for PAX8 expression using BC12 and compared to stainingpatterns using a rabbit polyclonal anti-PAX8 antibody (10336-1-AP,Proteintech). The mouse and rabbit PAX8 antibodies exhibited similarsensitivities for a variety of normal and neoplastic tissues. FIGS. 1-5show several examples of the PAX8 staining on normal and tumor tissuesfor kidney and ovarian examples.

The monoclonal mouse PAX8 antibody may offer distinct advantages withits improved specificity and even particularly its lack ofcross-reactivity with lymphocytes, pancreatic tissue (islets ofLangerhans), and neuroendocrine cells of the stomach. FIGS. 6, 7, 8, and9 show comparisons of the mouse monoclonal antibody [BC12] with therabbit polyclonal antibody, demonstrating the similarities in positivestaining for PAX8 by both antibodies, but the greater specificity ofBC12, which may not stain B-cells, as compared to the rabbit monoclonalantibody, where B-cell staining may be observed.

Additionally, the mouse monoclonal PAX8 antibody [BC12] may have anadvantage of specificity versus pancreatic tissue and certainneuroendocrine tumors such as carcinoids; whereas the rabbit polyclonalantibody may stain normal and neoplastic pancreatic tissues, and mayalso stain certain carcinoids. BC12 may not stain pancreatic tissue asmay be understood in FIGS. 10, 11, 12, and 13. Furthermore BC12 hasdemonstrated increased specificity on stomach tissue as well. The rabbitpolyclonal PAX8 antibody may stain a subset of cells in stomach tissuethat may not be stained by BC12 as can be understood in FIGS. 14 and 15.Cells stained in FIG. 15 stained positive by a chromogranin IHC assay;and thus, confirmed as neuroendocrine origin.

In some embodiments of the present invention, the mouse monoclonal PAX8antibody [BC12] may be suitable for use in many variations of the aboveprotocols and other methods known to those in the art. Specimens stainedwith BC12 may be archived using a permanent mounting media and acoverslip. The antibody [BC12] may also be used in an automated staininginstrument, using standard protocols. One can also envision the use ofmany alternative detection methods (e.g., fluorescence), detectionenzymes (e.g., alkaline phosphatase (AP), beta-galactosidase), andperhaps even chromogens (e.g., 3-amino-9-ethylcarbazole,5-bromo-4-chloro-3-indolyl phosphate, 3,3′,5,5′-tetramethylbenzidine,5-bromo-4-chloro-3-indolyl-(3-D-glucuronide), generally known to thosein the art.

The epitope for BC12 was shown to be included in the residues 258-272 ofPAX8, which is EQGLYPLPLLNSTLD. The epitope of the mouse monoclonal PAX8antibody, or a portion thereof, may be a useful antigen for theproduction of new monoclonal antibodies, including production in speciesother than mouse (e.g. rabbit, goat, horse, chicken, etc.).

While the use of BC12 in immunohistochemistry of formalin-fixed paraffinembedded tissues is described here, its utility in other immunoassaysmay be readily envisioned and are meant to be included in thisapplication. In particular, it may be well known that many of the samereagents used in IHC of FFPE may also be used in IHC of frozen-tissuesections. BC12 may also be useful in other immunoassays, includingELISA, perhaps using generally known methods.

In another aspect of the invention, perhaps related to IHC, a PAX8antibody may be used in conjunction with one or more additional primaryantibodies as part of a cocktail, to perform a “double-stain” procedure(also described as multi-stain or even multiplex). Such “double-stain”procedures may be generally well known in the art; however, the bestcombinations of primary antibodies for a particular diagnosticapplication may not be known.

In this method, a mouse monoclonal PAX8 antibody [BC12] could becombined with one or more antibodies in a single primary antibodycocktail. At least one of the additional antibodies could be derivedfrom a species perhaps even other than mouse such as a rabbit antibody.Species may include but is not limited to mouse, rabbit, goat, horse,chicken, human, any combinations thereof, or the like. In this manner,the multiple antibodies in the primary antibody cocktail may bedifferentiated in the subsequent detection and even visualization steps.For example, following incubation of the tissue sample with the primaryantibody cocktail, a cocktail of goat anti-mouse antibody conjugated toHRP and a goat anti-rabbit antibody conjugated to AP may be applied.Subsequently, chromogens specific for HRP and AP may be sequentiallyapplied. In this manner, two or more targets may be identified on thesame tissue sample with the resulting two colors. In this specificexample, mouse primary antibodies (including BC12) could result in brown(DAB) staining and rabbit primary antibodies could result in red (FastRed) staining. Multiplex MC may also employ primary antibodies from thesame host species, resulting the staining of the same color; however,the antibodies may be distinguished by different cellular localizationpatterns (cytoplasmic, membrane or nuclear).

Specifically, multiplex IHC may be performed by preparing FFPE tissuesfor staining in the usual manner, including dewaxing, hydration, andantigen retrieval. A cocktail of primary antibodies (in a buffereddiluent with carrier protein [e.g. BSA] and preservative [e.g. sodiumazide]) is applied to the tissue sample for a period of typically 30minutes. Importantly, the primary antibodies in the cocktail areisolated from two different host-species (e.g. mouse and rabbit). Forexample, a primary antibody cocktail containing mouse anti-p63 andrabbit anti-P504S is commonly used in prostate diagnosis. Secondaryantibodies conjugated to enzyme for detection (e.g. alkaline phosphatase[AP], horseradish peroxidase [HRP]) may then be applied to the tissuesample, typically for a period of 30 minutes. These secondary antibodyconjugates are typically raised in goat and bind the mouse or rabbit IgGof the primary antibodies previously applied. For example, a cocktail ofgoat anti-mouse-HRP and goat anti-rabbit-AP is often applied to themouse anti-p63 and rabbit anti-P504S described above. In this example,p63 (a mouse antibody) would be bound by goat anti-mouse-HRP and P504S(a rabbit antibody) would be bound by goat anti-rabbit-AP. The presenceand localization of both antibodies may then be distinguished by thesequential application of chromogens resulting in different colors.Specifically, each of the applied chromogens reacts with only one of thetwo detection enzymes (AP or HRP) to produce a colored stain at the siteof the antibody complex. In the p63+P504S example, 3,3′-diaminobenzidine(DAB) may produce a brown stain, catalyzed by the HRP of the p63antibody complex, while Fast Red (a diazonium salt and naphtholphosphate) may produce a red stain, catalyzed by the AP of the P504Santibody complex.

Multiplex IHC has several advantages over traditional single-stainmethods. For example, doubles-staining may take less time and use lessreagents than a single-stain method. The opportunity to visualizeperhaps by color results of two or more antigens in the same tissuesection may greatly ease the pathologist's interpretation.Double-staining also has the advantage of consuming less of a tissuesample (i.e. a single section), thus conserving precious tissue forother tests.

Multiple alternatives to a double-staining method are possible,including but not limited to the use of more than two antibodies, theuse of species other than mouse and rabbit, other chromogens anddetection systems, a different order of detection steps, the sequentialapplication of antibody regents instead of the use of cocktails, the useof goat anti-mouse-AP and goat anti-rabbit-HRP secondary antibodies, andperhaps even modifications resulting in three or more colors (which mayrequire a denaturing step).

An anti-PAX8 antibody, such as, but not limited to BC12, may be used inthe primary antibody cocktail of double-stain procedures in an methodthat may be useful for clinical diagnosis. For example, in cases where atumor of unknown origin is being investigated, or a differentialdiagnosis between a kidney cancer and another cancer is beingconsidered, the combination of PAX8 with another antibody may aid in thediagnosis.

An antibody cocktail may include a composition with at least twoantibodies or fragments thereof where at least one antibody bindsspecifically to PAX8 and at least one other antibody binds to an antigenincluding but not limited to: GATA-3, p63, PSA, ER, Mammaglobin,GCDFP-15, NKX3.1, Napsin A, TTF-1, CK20, CDX2, ERG, or the like, and anycombination thereof. Compositions may include combination such as butnot limited to PAX8 and GATA-3; PAX8 and p63; PAX8 and PSA; PAX8 and PSAand GATA-3; PAX8 and ER and Mammoglobin and GCDFP-15; PAX8 and ER; PAX8and Mammoglobin; PAX8 and GCDFP-15; PAX8 and NKX3.1; PAX8 and Napsin Aand TTF-1; PAX8 and Napsin A; PAX8 and TTF-1; PAX8 and CD20 and CDX2;PAX8 and CD20; PAX8 and CDX2; PAX8 and ERG and GATA-3; PAX8 and ERG; orthe like, or any combination thereof. The antibody cocktail may includeany antibody which specifically binds to PAX8; may be an antibody whichspecifically binds to PAX8 but not to B-cells; may be the BC12 antibodyor portions thereof, fragments thereof, or the like; may be an antibodywhich specifically binds to PAX8 but not to neuroendocrine cells,pancreatic cells, or any combination thereof; or the like.

Methods for antibody cocktails may include detecting at least twodifferent proteins in a biological sample, comprising the steps ofcontacting a biological sample (2) with a composition comprising atleast two antibodies or fragments thereof, wherein at least one of saidat least two antibodies or fragments thereof binds specifically to atleast PAX8, to form an antigen-antibody complex (3); and perhaps evendetecting said antigen-antibody complex.

Antibodies that may be useful for diagnosis when combined with a mousemonoclonal PAX8 antibody [BC12] in a primary antibody cocktail for usein multi-stain procedures may include:

Diagnosis of tumor of unknown Antibody Cocktail origin or differentialdiagnosis PAX8 + GATA-3 Renal vs Bladder or Breast PAX8 + p63 Renal vsRenal Pelvis PAX8 + PSA + GATA-3 Renal vs Prostate vs Bladder PAX8 + ERand/or Mammaglobin Renal vs Breast and/or GCDFP-15 PAX8 + NKX3.1 Renalvs Prostate PAX8 + Napsin A and/or TTF-1 Renal vs. Lung PAX8 + CK20and/orCDX2 Renal vs. Colon PAX8 + ERG + GATA-3 Renal vs Prostate vsBladder

Examples of antibody cocktails containing PAX8 are described below. Insome cases, example cocktails were stained on normal tissues where theantigen is known to be present in both normal and neoplastic tissues.

PAX8+GATA-3: A double-stain IHC primary antibody cocktail containingmouse monoclonal PAX8+rabbit monoclonal GATA-3 may be useful fordiagnosis, including for the discrimination of renal carcinomas (PAX8)from breast or urothelial carcinomas (GATA-3).

PAX8+p63: p53 homologue p63 encodes for different isotypes able toeither transactivate p53 reporter genes (TAp63) or act asp53-dominant-negatives. p63 is detected in prostatic basal cells innormal prostate, however, it is negative in malignant tumors of theprostate gland. Thus, p63 is a useful differential marker for benign andmalignant tumors of the prostate gland. p63 is also a marker ofmyoepithelial cells in breast ducts and may be useful in identifyingbreast carcinoma. As a marker or urothelial carcinoma, p63 may alsostain renal pelvis urothelial carcinoma. A double-stain of PAX8+p63 maybe useful in the differential diagnosis of carcinoma of the renal pelvisfrom renal cell carcinoma.

FIGS. 16 and 17 show an example of multiplex IHC staining of mousemonoclonal PAX8 [BC12] +rabbit monoclonal p63 [EPR5701] (Cell Signaling)on renal cell carcinoma (FIG. 16) and normal bladder (FIG. 17). Adetection system of goat anti-mouse-HRP+goat anti-rabbit-AP with DAB andFast Red chromogens, resulting in brown (PAX8) or red (p63) staining.

PAX8+PSA+GATA-3: PSA is a chymotrypsin-like serine protease produced bythe prostate epithelium. PSA is used to confirm prostatic acinar cellorigin in primary and metastatic carcinomas and to rule outnon-prostatic carcinoma mimics. A triple-stain of PAX8+PSA+GATA-3 may beuseful in the differential diagnosis of renal cell carcinoma, prostateadenocarcinoma and urothelial carcinoma.

GATA-3 (GATA binding protein 3) is a member of the GATA family oftranscription factors. Among several other roles, GATA-3 has is as a keyplayer in luminal cell differentiation in the mammary gland. Theexpression of GATA-3 has a strong association with the expression ofestrogen receptor-alpha (ER) in breast cancer, and there is mountingevidence that GATA-3 can be used as a clinical marker to determineresponse to hormonal therapy and to refine the prognosis of breastcancer patients. GATA-3 has also been shown to be a novel marker forbladder cancer. In one study, GATA-3 stained 67% of 308 urothelialcarcinomas, but none for prostate or renal carcinomas. A double-stain ofPAX8+PSA+GATA-3 may be useful in the differential diagnosis of renalcell carcinoma, prostate adenocarcinoma and urothelial carcinoma, seeFIGS. 18, 19, and 20.

FIGS. 18, 19, and 20 show an example of multiplex IHC staining of mousemonoclonal PAX8 [BC12] +rabbit monoclonal PSA [EP1588Y] (e.g., BiocareMedical)+rabbit monoclonal GATA-3 [D13C9] (Cell Signaling) on renal cellcarcinoma (FIG. 18), prostate adenocarcinoma (FIG. 19) and urothelialcarcinoma (FIG. 20). A detection system of goat anti-mouse-HRP+goatanti-rabbit-AP with DAB and Fast Red chromogens, resulting in nuclearbrown (PAX8), cytoplasmic red (PSA) or nuclear red (GATA-3) staining.

PAX8+ER+Mammaglobin: Estrogen receptor alpha (ER) is a nucleartranscription factor and a member of the steroid hormone receptorfamily. ER is routinely used in the diagnosis, prognosis and predictionof response to hormonal therapy for breast cancer patients. Mammaglobinis a mammary-specific member of the uteroglobin family and is known tobe overexpressed in human breast cancer. In normal breast tissue,mammaglobin labels breast ductal and lobular epithelial cells. However,mammaglobin is expressed in a higher percentage of lobular carcinomaversus ductal cell carcnimoma. A double-stain or perhaps even atriple-stain of PAX8+ER+mammaglobin may be useful in the diganosis ofrenal cell carcinoma versus breast carcinoma. FIGS. 21 and 22 showstaining of the PAX8+ER+mammaglobin cocktail.

FIGS. 21 and 22 show multiplex IHC staining of a mouse monoclonal PAX8[BC12] +rabbit monoclonal ER [SP1] (Biocare Medical)+rabbit monoclonalMammaglobin [31-A5] (Zeta) on renal cell carcinoma (FIG. 21) and normalbreast (FIG. 22). A detection system of goat anti-mouse-HRP+goatanti-rabbit-AP with DAB and Fast Red chromogens, resulting in nuclearbrown (PAX8), cytoplasmic red (Mammaglobin) or nuclear red (ER)staining.

PAX8+NKX3.1: The homeodomain containing transcription factor NKX3.1 is aputative prostate tumor suppressor that is expressed in a largelyprostate-specific and androgen-regulated manner. NKX3.1 protein has beenfound to be positive in the vast majority of primary pro staticadenocarcinomas. The sensitivity for identifying metastatic prostaticadenocarcinomas overall was 98.6% (68/69 cases positive) for NKX3.1.NKX3.1 stains nuclei in both normal and prostate cancer. A double-stainof PAX8+NKX3.1 may be useful in the differential diagnosis of renal cellcarcinoma from prostate adenocarcinoma, see FIGS. 23 and 24.

FIGS. 23 and 24 show multiplex IHC staining of mouse monoclonal PAX8[BC12] +rabbit polyclonal NKX3.1 (e.g., Biocare Medical, CP422) on renalcell carcinoma (FIG. 23) and prostate adenocarcinoma (FIG. 24). Adetection system of goat anti-mouse-HRP+goat anti-rabbit-AP with DAB andFast Red chromogens, resulting in brown (PAX8) or red (NKX3.1) staining.

PAX8+Napsin A: Napsin A is an aspartic detected in the cytoplasm of type2 pneumocytes and alveolar macrophages. Napsin A is a sensitive andspecific marker for lung adenocarcinomas. In one study, Napsin Aidentified 70 of 83 (84%) lung adenocarcinomas. A double-stain ofPAX8+Nap sin A may be useful in the differential diagnosis of renal cellcarcinoma from lung adenocarcinoma. FIGS. 25 and 25 show staining of aPAX8+Napsin A antibody cocktail.

FIGS. 25 and 26 show multiplex IHC staining of mouse monoclonal PAX8[BC12] +rabbit polyclonal Napsin A (Biocare Medical, PP434) on renalcell carcinoma (FIG. 25) and normal lung (FIG. 26). A detection systemof goat anti-mouse-HRP+goat anti-rabbit-AP with DAB and Fast Redchromogens, resulting in brown (PAX8) or red (Napsin A) staining.

PAX8+CK20: Cytokeratin 20 (CK20) is a 46 kDa intermediate filamentprotein that is a useful marker in the identification of colonadenocarcinoma. A double-stain of PAX8+CK20 may be useful in thedifferential diagnosis of renal cell carcinoma from colonadenocarcinoma. FIGS. 27 and 28 show staining of a PAX8+CK20 antibodycocktail.

FIGS. 27 and 28 show multiplex IHC staining of mouse monoclonal PAX8[BC12] +rabbit monoclonal CK20 [EP23] (Epitomics) on renal cellcarcinoma (FIG. 27) and normal colon (FIG. 28). A detection system ofgoat anti-mouse-HRP+goat anti-rabbit-AP with DAB and Fast Redchromogens, resulting in brown (PAX8) or red (CK20) staining.

PAX8+ERG+GATA-3: In human prostate cancer, the ERG oncogene isfrequently overexpressed due to chromosomal translocations involving ERGand regulatory sequences of the TMPRSS2 or other androgen responsivegenes. Strong concordance between the TMPRSS2-ERG translocation anddetection of the protein product by IHC has been demonstrated. Anti-ERGantibodies are specific markers for prostate adenocarcinoma. Adouble-stain of PAX8+ERG may be useful in the differential diagnosis ofrenal cell carcinoma from prostatic adenocarcinoma. A double-stain ofPAX8+ERG+GATA-3 may be useful in the differential diagnosis of renalcell carcinoma from prostatic adenocarcinoma and urothelial carcinoma,see FIGS. 29, 30, and 31.

FIGS. 29, 30, and 31 show multiplex MC staining of mouse monoclonal PAX8[BC12] +rabbit monoclonal ERG [ER3863] (Epitomics)+rabbit monoclonalGATA-3 [D13C9] (Cell Signaling) on renal cell carcinoma (FIG. 29),prostatic adenocarcinoma (FIG. 30) and urothelial carcinoma (FIG. 31). Adetection system of goat anti-mouse-HRP+goat anti-rabbit-AP with DAB andFast Red chromogens, resulting in brown (PAX8), or red (ERG or GATA-3)staining. ERG and GATA-3 staining may be distinguished by morphology.(Note that normal endothelial cells present in most tissues are known tostain with ERG.)

The mouse monoclonal PAX8 antibody [BC12] may be specific for detectionof PAX8 and may be useful in immunohistochemical procedures fordiagnosis of several types of cancers in human tissue samples. Inparticular, BC12, nucleic acid sequences SEQ ID NO: 1 and/or SEQ ID NO:2, and amino acid SEQ ID NO: 3 can be used and have advantages overpreviously known PAX8 antibodies, including greater specificity versusB-cells, as well as a lack of cross-reactivity and staining ofpancreatic tissues and neuroendocrine cells of the stomach.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. It involvesboth antibody techniques as well as devices to accomplish theappropriate antibody. In this application, the antibody techniques aredisclosed as part of the results shown to be achieved by the variousdevices described and as steps which are inherent to utilization. Theyare simply the natural result of utilizing the devices as intended anddescribed. In addition, while some devices are disclosed, it should beunderstood that these not only accomplish certain methods but also canbe varied in a number of ways. Importantly, as to all of the foregoing,all of these facets should be understood to be encompassed by thisdisclosure.

The discussion included in this application is intended to serve as abasic description. The reader should be aware that the specificdiscussion may not explicitly describe all embodiments possible; manyalternatives are implicit. It also may not fully explain the genericnature of the invention and may not explicitly show how each feature orelement can actually be representative of a broader function or of agreat variety of alternative or equivalent elements. Again, these areimplicitly included in this disclosure. Where the invention is describedin device-oriented terminology, each element of the device implicitlyperforms a function. Apparatus claims may not only be included for thedevice described, but also method or process claims may be included toaddress the functions the invention and each element performs. Neitherthe description nor the terminology is intended to limit the scope ofthe claims that will be included in any subsequent patent application.

It should also be understood that a variety of changes may be madewithout departing from the essence of the invention. Such changes arealso implicitly included in the description. They still fall within thescope of this invention. A broad disclosure encompassing the explicitembodiment(s) shown, the great variety of implicit alternativeembodiments, and the broad methods or processes and the like areencompassed by this disclosure and may be relied upon when drafting theclaims for any subsequent patent application. It should be understoodthat such language changes and broader or more detailed claiming may beaccomplished at a later date (such as by any required deadline) or inthe event the applicant subsequently seeks a patent filing based on thisfiling. With this understanding, the reader should be aware that thisdisclosure is to be understood to support any subsequently filed patentapplication that may seek examination of as broad a base of claims asdeemed within the applicant's right and may be designed to yield apatent covering numerous aspects of the invention both independently andas an overall system.

Further, each of the various elements of the invention and claims mayalso be achieved in a variety of manners. Additionally, when used orimplied, an element is to be understood as encompassing individual aswell as plural structures that may or may not be physically connected.This disclosure should be understood to encompass each such variation,be it a variation of an embodiment of any apparatus embodiment, a methodor process embodiment, or even merely a variation of any element ofthese. Particularly, it should be understood that as the disclosurerelates to elements of the invention, the words for each element may beexpressed by equivalent apparatus terms or method terms—even if only thefunction or result is the same. Such equivalent, broader, or even moregeneric terms should be considered to be encompassed in the descriptionof each element or action. Such terms can be substituted where desiredto make explicit the implicitly broad coverage to which this inventionis entitled. As but one example, it should be understood that allactions may be expressed as a means for taking that action or as anelement which causes that action. Similarly, each physical elementdisclosed should be understood to encompass a disclosure of the actionwhich that physical element facilitates. Regarding this last aspect, asbut one example, the disclosure of a “detection” or “detector” should beunderstood to encompass disclosure of the act of “detecting” —whetherexplicitly discussed or not—and, conversely, were there effectivelydisclosure of the act of “detecting”, such a disclosure should beunderstood to encompass disclosure of a “detector” and even a “means fordetecting.” Such changes and alternative terms are to be understood tobe explicitly included in the description. Further, each such means(whether explicitly so described or not) should be understood asencompassing all elements that can perform the given function, and alldescriptions of elements that perform a described function should beunderstood as a non-limiting example of means for performing thatfunction.

Any law, statutes, regulations, or rules mentioned in this applicationfor patent; or patents, publications, or other references mentioned inthis application for patent are hereby incorporated by reference. Anypriority case(s) claimed by this application is hereby appended andhereby incorporated by reference. In addition, as to each term used itshould be understood that unless its utilization in this application isinconsistent with a broadly supporting interpretation, common dictionarydefinitions should be understood as incorporated for each term and alldefinitions, alternative terms, and synonyms such as contained in theRandom House Webster's Unabridged Dictionary, second edition are herebyincorporated by reference. Finally, all references listed in any list ofReferences or other information statement filed with the application arehereby appended and hereby incorporated by reference, however, as toeach of the above, to the extent that such information or statementsincorporated by reference might be considered inconsistent with thepatenting of this/these invention(s) such statements are expressly notto be considered as made by the applicant(s).

Thus, the applicant(s) should be understood to have support to claim andmake a statement of invention to at least: i) each of the antibodydevices as herein disclosed and described, ii) the related methodsdisclosed and described, iii) similar, equivalent, and even implicitvariations of each of these devices and methods, iv) those alternativedesigns which accomplish each of the functions shown as are disclosedand described, v) those alternative designs and methods which accomplisheach of the functions shown as are implicit to accomplish that which isdisclosed and described, vi) each feature, component, and step shown asseparate and independent inventions, vii) the applications enhanced bythe various systems or components disclosed, viii) the resultingproducts produced by such systems or components, ix) each system,method, and element shown or described as now applied to any specificfield or devices mentioned, x) methods and apparatuses substantially asdescribed hereinbefore and with reference to any of the accompanyingexamples, xi) an apparatus for performing the methods described hereincomprising means for performing the steps, xii) the various combinationsand permutations of each of the elements disclosed, xiii) eachpotentially dependent claim or concept as a dependency on each and everyone of the independent claims or concepts presented, and xiv) allinventions described herein.

With regard to claims whether now or later presented for examination, itshould be understood that for practical reasons and so as to avoid greatexpansion of the examination burden, the applicant may at any timepresent only initial claims or perhaps only initial claims with onlyinitial dependencies. The office and any third persons interested inpotential scope of this or subsequent applications should understandthat broader claims may be presented at a later date in this case, in acase claiming the benefit of this case, or in any continuation in spiteof any preliminary amendments, other amendments, claim language, orarguments presented, thus throughout the pendency of any case there isno intention to disclaim or surrender any potential subject matter. Itshould be understood that if or when broader claims are presented, suchmay require that any relevant prior art that may have been considered atany prior time may need to be re-visited since it is possible that tothe extent any amendments, claim language, or arguments presented inthis or any subsequent application are considered as made to avoid suchprior art, such reasons may be eliminated by later presented claims orthe like. Both the examiner and any person otherwise interested inexisting or later potential coverage, or considering if there has at anytime been any possibility of an indication of disclaimer or surrender ofpotential coverage, should be aware that no such surrender or disclaimeris ever intended or ever exists in this or any subsequent application.Limitations such as arose in Hakim v. Cannon Avent Group, PLC, 479 F.3d1313 (Fed. Cir 2007), or the like are expressly not intended in this orany subsequent related matter. In addition, support should be understoodto exist to the degree required under new matter laws—including but notlimited to European Patent Convention Article 123(2) and United StatesPatent Law 35 USC 132 or other such laws—to permit the addition of anyof the various dependencies or other elements presented under oneindependent claim or concept as dependencies or elements under any otherindependent claim or concept. In drafting any claims at any time whetherin this application or in any subsequent application, it should also beunderstood that the applicant has intended to capture as full and broada scope of coverage as legally available. To the extent thatinsubstantial substitutes are made, to the extent that the applicant didnot in fact draft any claim so as to literally encompass any particularembodiment, and to the extent otherwise applicable, the applicant shouldnot be understood to have in any way intended to or actuallyrelinquished such coverage as the applicant simply may not have beenable to anticipate all eventualities; one skilled in the art, should notbe reasonably expected to have drafted a claim that would have literallyencompassed such alternative embodiments.

Further, if or when used, the use of the transitional phrase“comprising” is used to maintain the “open-end” claims herein, accordingto traditional claim interpretation. Thus, unless the context requiresotherwise, it should be understood that the term “comprise” orvariations such as “comprises” or “comprising”, are intended to implythe inclusion of a stated element or step or group of elements or stepsbut not the exclusion of any other element or step or group of elementsor steps. Such terms should be interpreted in their most expansive formso as to afford the applicant the broadest coverage legally permissible.The use of the phrase, “or any other claim” is used to provide supportfor any claim to be dependent on any other claim, such as anotherdependent claim, another independent claim, a previously listed claim, asubsequently listed claim, and the like. As one clarifying example, if aclaim were dependent “on claim 20 or any other claim” or the like, itcould be re-drafted as dependent on claim 1, claim 15, or even claim 25(if such were to exist) if desired and still fall with the disclosure.It should be understood that this phrase also provides support for anycombination of elements in the claims and even incorporates any desiredproper antecedent basis for certain claim combinations such as withcombinations of method, apparatus, process, and the like claims.

Finally, any claims set forth at any time are hereby incorporated byreference as part of this description of the invention, and theapplicant expressly reserves the right to use all of or a portion ofsuch incorporated content of such claims as additional description tosupport any of or all of the claims or any element or component thereof,and the applicant further expressly reserves the right to move anyportion of or all of the incorporated content of such claims or anyelement or component thereof from the description into the claims orvice-versa as necessary to define the matter for which protection issought by this application or by any subsequent continuation, division,or continuation-in-part application thereof, or to obtain any benefitof, reduction in fees pursuant to, or to comply with the patent laws,rules, or regulations of any country or treaty, and such contentincorporated by reference shall survive during the entire pendency ofthis application including any subsequent continuation, division, orcontinuation-in-part application thereof or any reissue or extensionthereon.

1-23. (canceled)
 24. An antibody or fragment thereof that bindsspecifically to PAX8 comprising heavy chain variable regioncomplementarity determining region (CDR) sequences set forth as SEQ IDNOs: 4, 5, and 6; and light chain variable region CDR sequences setforth as SEQ ID NOs: 7, 8 and
 9. 25. An antibody or fragment thereofaccording to claim 24 that specifically binds to at least onepolypeptide with an amino acid sequence comprising residues of SEQ IDNO:
 3. 26. An antibody or fragment thereof according to claim 24 whereinsaid antibody or said fragment thereof comprises a light chain variableregion comprising the amino acid sequence encoded by the nucleic acidsequence of SEQ ID NO: 2 and a heavy chain variable region comprisingthe amino acid sequence encoded by the nucleic acid sequence of SEQ IDNO:
 1. 27. An antibody or fragment thereof according to claim 24 whereinsaid antibody or said fragment thereof which binds specifically to atleast PAX8 comprises an antibody or fragment thereof which bindsspecifically to at least PAX8 and does not bind to B-cells.
 28. Acomposition comprising at least two antibodies or fragments thereof,wherein said at least one of said at least two antibodies or fragmentsthereof comprises said antibody or said fragments thereof having the CDRregions according to claim
 24. 29. A composition according to claim 28wherein said at least one other of said at least two antibodies orfragments thereof binds specifically to an antigen selected from a groupconsisting of GATA-3, p63, PSA, ER, Mammaglobin, GCDFP-15, NKX3.1,Napsin A, TTF-1, CK20, CDX2, ERG, and any combination thereof.
 30. Acomposition according to claim 28 wherein said at least two antibodiesor fragments thereof each bind specifically to proteins selected from agroup consisting of: PAX8 and GATA-3; PAX8 and p63; PAX8 and PSA; PAX8and PSA and GATA-3; PAX8 and ER and Mammoglobin and GCDFP-15; PAX8 andER; PAX8 and Mammoglobin; PAX8 and GCDFP-15; PAX8 and NKX3.1; PAX8 andNapsin A and TTF-1; PAX8 and Napsin A; PAX8 and TTF-1; PAX8 and CD20 andCDX2; PAX8 and CD20; PAX8 and CDX2; PAX8 and ERG and GATA-3; and PAX8and ERG.
 31. A composition according to claim 28 wherein said at leasttwo antibodies or fragments thereof are derived from at least twodifferent species.
 32. A composition according to claim 31 wherein saidat least two different species is selected from a group consisting ofmouse, rabbit, goat, horse, chicken, human, and any combination thereof.33. A composition according to claim 28 wherein said at least twoantibodies or fragments thereof comprises a double-stain procedure. 34.A composition according to claim 28 wherein said at least two antibodiesor fragments thereof are capable of providing different visualizationresults.
 35. A composition according to claim 34 wherein saidvisualization results comprises color results.
 36. An antibody accordingto claim 24 wherein said antibody comprises a monoclonal antibody. 37.An antibody according to claim 36 wherein said monoclonal antibody isselected from a group consisting of a mouse monoclonal antibody, arabbit monoclonal antibody, a goat monoclonal antibody, a horsemonoclonal antibody, a chicken monoclonal antibody, a humanizedmonoclonal antibody, a chimeric antibody, and any combination thereof.38. An antibody according to claim 24 wherein said antibody comprises anisolated antibody.
 39. An antibody according to claim 24 wherein saidfragment thereof comprises an antigen binding fragment thereof.
 40. Anantibody according to claim 24 and further comprising a label attachedto said antibody or fragment thereof.
 41. An antibody according to claim40 wherein said label is selected from a group consisting of aradioactive element, magnetic particles, radioisotope, fluorescent dye,enzyme, toxin, signal, stain, and any combination thereof.
 42. A methodfor detecting PAX8 in a biological sample comprising the steps of:contacting a biological sample with said antibody or fragment thereofaccording to claim 24; and detecting binding of said antibody with anantigen in said biological sample using said antibody detection element.43. A method according to claim 42 wherein said biological sample isselected from a group consisting of a normal tissue, neoplastic tissue,kidney tissue, ovarian tissue, thyroid tissue, endometrial tissue, renaltissue, tonsil tissue, pancreas tissue, colon tissue, lymph node tissue,neoplastic pancreatic tissue, stomach tissue, bladder tissue, prostatetissue, lung tissue and breast tissue.
 44. A method according to claim42 wherein said detecting comprises a method selected from a groupconsisting of immunohistochemistry (IHC), IHC of FFPE, ICH offrozen-tissue sections, and ELISA.
 45. An antibody or fragment thereofaccording to claim 24 wherein said antibody or said fragment thereofcomprises a light chain variable region comprising an amino acidsequence at least about 90% identical to an amino acid sequence encodedby a nucleic acid sequence of SEQ ID NO:
 2. 46. An antibody or fragmentthereof according to claim 24 wherein said antibody or said fragmentthereof comprises a heavy chain variable region comprising an amino acidsequence at least about 90% identical to an amino acid sequence encodedby the nucleic acid sequence of SEQ ID NO: 1.